Many VA patients are susceptible and succumb to infections with the opportunistic pathogen, Pseudomonas aeruginosa. This can occur as a complication of emphysema, chronic bronchitis, cancer and immunosuppressive drug therapy. Exposure to this ubiquitous bacterium can result in nosocomial infections, which are common via respiratory ventilators, catheters, lumbar punctures and general surgery. P. aeruginosa is highly tolerant or resistant to most antibiotics, making it difficult to control such infections, which leads to a high mortality rate. The goal of tis research is to improve our understanding of the biosynthesis of a protective capsule-like polysaccharide called alginate, which is produced as a virulence factor by P. aeruginosa. During chronic respiratory infections (e.g., COPD), adaptive mutations occur in vivo that lead to the over production of this exopolysaccharide, which confers resistance to phagocytic killing. Such clinical isolates demonstrate mucoid colony morphology. Improving our understanding of this pathogenic mechanism in P. aeruginosa will enhance the management of pulmonary disease caused by this bacterium. Most of the enzymes for the production of alginate are clustered in an operon of 12 genes. In this study, we will: (1) Determine the role of the second messenger cyclic di-GMP in the polymerization of alginate, (2) Characterize the role of the membrane fusion protein (MFP) domain in Alg44, and (3) Characterize the protein-protein interactions among the alginate secretion proteins. In addition, as part of a drug discovery plan, we will (4) screen smal compound libraries for those that antagonize the acetylation of alginate, which is required for resistance to phagocytosis. In collaboration with a structural biologist, we will determine the structures of all the alginate biosynthetic proteins, which will lead to the analysis of site-direced mutants to better understand the polymerization-secretion complex. The long-term goal of this research is to understand the functions of all of the components required for the biosynthesis of alginate, which is now recognized as a critical virulence factor during pulmonary infection. The information gained could be vital for the development of new therapeutic approaches in the treatment of P. aeruginosa infections. The results of these studies will also contribute to our overall understanding of bacterial capsule biosynthesis, which is a common mechanism of bacterial virulence for avoiding the host immune response.